Insect inhibitory proteins

ABSTRACT

Pesticidal proteins exhibiting toxic activity against Lepidopteran pest species are disclosed, and include, but are not limited to, TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, and TIC7473PL. DNA constructs are provided which contain a recombinant nucleic acid sequence encoding one or more of the disclosed pesticidal proteins. Transgenic plants, plant cells, seed, and plant parts resistant to Lepidopteran infestation are provided which contain recombinant nucleic acid sequences encoding the pesticidal proteins of the present invention. Methods for detecting the presence of the recombinant nucleic acid sequences or the proteins of the present invention in a biological sample, and methods of controlling Lepidopteran species pests using any of the TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, and TIC7473PL pesticidal proteins are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/247,500, filed Aug. 25, 2016, which claims the benefit of U.S.Provisional Application No. 62/210,737, filed Aug. 27, 2015, each ofwhich are incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The file named “P34464US02_SEQ.TXT” containing a computer-readable formof the Sequence Listing was created on Nov. 1, 2018. This file is 94,544bytes (measured in MS-Windows®), filed contemporaneously by electronicsubmission (using the United States Patent Office EFS-Web filingsystem), and incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to the field of insect inhibitoryproteins. A novel class of proteins exhibiting insect inhibitoryactivity against agriculturally-relevant pests of crop plants and seedsare disclosed. In particular, the disclosed class of proteins isinsecticidally active against agriculturally-relevant pests of cropplants and seeds, particularly Lepidopteran species of insect pests.Plants, plant parts, and seeds containing a recombinant polynucleotideconstruct encoding one or more of the disclosed toxin proteins areprovided.

BACKGROUND OF THE INVENTION

Improving crop yield from agriculturally significant plants including,among others, corn, soybean, sugarcane, rice, wheat, vegetables, andcotton, has become increasingly important. In addition to the growingneed for agricultural products to feed, clothe and provide energy for agrowing human population, climate-related effects and pressure from thegrowing population to use land other than for agricultural practices arepredicted to reduce the amount of arable land available for farming.These factors have led to grim forecasts of food security, particularlyin the absence of major improvements in plant biotechnology andagronomic practices. In light of these pressures, environmentallysustainable improvements in technology, agricultural techniques, andpest management are vital tools to expand crop production on the limitedamount of arable land available for farming.

Insects, particularly insects within the order Lepidoptera andColeoptera, are considered a major cause of damage to field crops,thereby decreasing crop yields over infested areas. Lepidopteran pestspecies which negatively impact agriculture include, but are not limitedto, Black armyworm (Spodoptera exempta), Black cutworm (Agrotis Cornearworm (Helicoverpa zea), Cotton leaf worm (Alabama argillacea),Diamondback moth (Plutella xylostella), European corn borer (Ostrinianubilalis), Fall armyworm (Spodoptera frugiperda), Cry1Fa1 resistantFall armyworm (Spodoptera frugiperda), Old World bollworm (OWB,Helicoverpa armigera), Southern armyworm (Spodoptera eridania), Soybeanlooper (Chrysodeixis includens), Spotted bollworm (Earias vittella),Southwestern corn borer (Diatraea grandiosella), Tobacco budworm(Heliothis virescens), Tobacco cutworm (Spodoptera litura, also known ascluster caterpillar), Western bean cutworm (Striacosta albicosta), andVelvet bean caterpillar (Anticarsia gemmatalis).

Historically, the intensive application of synthetic chemicalinsecticides was relied upon as the pest control agent in agriculture.Concerns for the environment and human health, in addition to emergingresistance issues, stimulated the research and development of biologicalpesticides. This research effort led to the progressive discovery anduse of various entomopathogenic microbial species, including bacteria.

The biological control paradigm shifted when the potential ofentomopathogenic bacteria, especially bacteria belonging to the genusBacillus, was discovered and developed as a biological pest controlagent. Strains of the bacterium Bacillus thuringiensis (Bt) have beenused as a source for pesticidal proteins since it was discovered that Btstrains show a high toxicity against specific insects. Bt strains areknown to produce delta-endotoxins that are localized within parasporalcrystalline inclusion bodies at the onset of sporulation and during thestationary growth phase (e.g., Cry proteins), and are also known toproduce secreted insecticidal protein. Upon ingestion by a susceptibleinsect, delta-endotoxins as well as secreted toxins exert their effectsat the surface of the midgut epithelium, disrupting the cell membrane,leading to cell disruption and death. Genes encoding insecticidalproteins have also been identified in bacterial species other than Bt,including other Bacillus and a diversity of additional bacterialspecies, such as Brevibacillus laterosporus, Lysinibacillus sphaericus(“Ls” formerly known as Bacillus sphaericus) and Paenibacilluspopilliae.

Crystalline and secreted soluble insecticidal toxins are highly specificfor their hosts and have gained worldwide acceptance as alternatives tochemical insecticides. For example, insecticidal toxin proteins havebeen employed in various agricultural applications to protectagriculturally important plants from insect infestations, decrease theneed for chemical pesticide applications, and increase yields.Insecticidal toxin proteins are used to control agriculturally-relevantpests of crop plants by mechanical methods, such as spraying to dispersemicrobial formulations containing various bacteria strains onto plantsurfaces, and by using genetic transformation techniques to producetransgenic plants and seeds expressing insecticidal toxin protein.

The use of transgenic plants expressing insecticidal toxin proteins hasbeen globally adapted. For example, in 2012, 26.1 million hectares wereplanted with transgenic crops expressing Bt toxins (James, C., GlobalStatus of Commercialized Biotech/GM Crops: 2012. ISAAA Brief No. 44).The global use of transgenic insect-protected crops and the limitednumber of insecticidal toxin proteins used in these crops has created aselection pressure for existing insect alleles that impart resistance tothe currently-utilized insecticidal proteins.

The development of resistance in target pests to insecticidal toxinproteins creates the continuing need for discovery and development ofnew forms of insecticidal toxin proteins that are useful for managingthe increase in insect resistance to transgenic crops expressinginsecticidal toxin proteins. New protein toxins with improved efficacyand which exhibit control over a broader spectrum of susceptible insectspecies will reduce the number of surviving insects which can developresistance alleles. In addition, the use in one plant of two or moretransgenic insecticidal toxin proteins toxic to the same insect pest anddisplaying different modes of action reduces the probability ofresistance in any single target insect species.

Thus, the inventors disclose herein a novel protein toxin family fromPaenibacillus popilliae, along with similar toxin proteins, variantproteins, and exemplary recombinant proteins that exhibit insecticidalactivity against target Lepidopteran species, particularly against Blackarmyworm (Spodoptera exempta), Black cutworm (Agrotis ipsilon), Cornearworm (Helicoverpa zea), Cotton leaf worm (Alabama argillacea),Diamondback moth (Plutella xylostella), European corn borer (Ostrinianubilalis), Fall armyworm (Spodoptera frugiperda), Cry1Fa1 resistantFall armyworm (Spodoptera frugiperda), Old World bollworm (OWB,Helicoverpa armigera), Southern armyworm (Spodoptera eridania), Soybeanlooper (Chrysodeixis includens), Spotted bollworm (Earias vittella),Southwestern corn borer (Diatraea grandiosella), Tobacco budworm(Heliothis virescens), Tobacco cutworm (Spodoptera litura, also known ascluster caterpillar), Western bean cutworm (Striacosta albicosta), andVelvet bean caterpillar (Anticarsia gemmatalis).

SUMMARY OF THE INVENTION

Disclosed herein is a novel group of pesticidal proteins with insectinhibitory activity (toxin proteins), referred to herein as TIC6757,TIC7472, and TIC7473 belonging to the TIC6757 protein toxin class, whichare shown to exhibit inhibitory activity against one or more pests ofcrop plants. The TIC6757 protein and proteins in the TIC6757 proteintoxin class can be used alone or in combination with other insecticidalproteins and toxic agents in formulations and in planta, thus providingalternatives to insecticidal proteins and insecticide chemistriescurrently in use in agricultural systems.

In one embodiment, disclosed in this application is a recombinantnucleic acid molecule comprising a heterologous promoter fragmentoperably linked to a polynucleotide segment encoding a pesticidalprotein or fragment thereof, wherein (a) said pesticidal proteincomprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, or SEQ ID NO:18; or (b) said pesticidal protein comprises anamino acid sequence having at least 85%, or 90%, or 95%, or 98%, or 99%,or about 100% amino acid sequence identity to SEQ ID NO:4, SEQ ID NO:2,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, or SEQ ID NO:18; or (c) said polynucleotide segment hybridizesto a polynucleotide having the nucleotide sequence of SEQ ID NO:3, SEQID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, and SEQ ID NO:17; or (d) said polynucleotidesegment encoding a pesticidal protein or fragment thereof comprises apolynucleotide sequence having at least 65%, or 70%, or 75%, or 80%, or85%, or 90%, or 95%, or 98%, or 99%, or about 100% sequence identity tothe nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ IDNO:17; or (e) said recombinant nucleic acid molecule is in operablelinkage with a vector, and said vector is selected from the groupconsisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial oryeast artificial chromosome. The recombinant nucleic acid molecule cancomprise a sequence that functions to express the pesticidal protein ina plant; or is expressed in a plant cell to produce a pesticidallyeffective amount of pesticidal protein.

In another embodiment of this application are host cells comprising arecombinant nucleic acid molecule of the application, wherein the hostcell is selected from the group consisting of a bacterial and a plantcell. Contemplated bacterial host cells include Agrobacterium,Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas,Klebsiella, Pantoec, and Erwinia. In certain embodiments, said Bacillusspecies is Bacillus cereus or Bacillus thuringiensis, said Brevibacillusis Brevibacillus laterosperous, or Escherichia is Escherichia coli.Contemplated plant host cells include a dicotyledonous plant cell and amonocotyledonous plant cell. Contemplated plant cells further include analfalfa, banana, barley, bean, broccoli, cabbage, brassica, carrot,cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus,coconut, coffee, corn, clover, cotton (Gossypium sp.), a cucurbit,cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops,leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion,ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine,potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice,rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetcorn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,triticale, turf grass, watermelon, and wheat plant cell.

In another embodiment, the pesticidal protein exhibits activity againstLepidopteran insects, including Velvet bean caterpillar, Sugarcaneborer, Lesser cornstalk borer, Corn earworm, Tobacco budworm, Soybeanlooper, Black armyworm, Southern armyworm, Fall armyworm, Beet armyworm,Old World bollworm, Oriental leaf worm, Pink bollworm, Black cutworm,Southwestern Corn Borer, Cotton leaf worm, Diamond back moth, Spottedbowl worm, Tobacco cut worm, Western bean cutworm, and European cornborer.

Also contemplated in this application are plants comprising arecombinant nucleic acid molecule comprising a heterologous promoterfragment operably linked to a polynucleotide segment encoding apesticidal protein or fragment thereof, wherein: (a) said pesticidalprotein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18; or (b) saidpesticidal protein comprises an amino acid sequence having at least 85%,or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequenceidentity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:12, SEQ IDNO:16, or SEQ ID NO:18; or (c) said polynucleotide segment hybridizesunder stringent hybridization conditions to the compliment of thenucleotide sequence of SEQ ID NO:3, SEQ ID NO:15, or SEQ ID NO:17; or(d) said plant exhibits a detectable amount of said pesticidal protein.In certain embodiments, the pesticidal protein comprises SEQ ID NO:4,SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18.In one embodiment, the plant is either a dicotyledonous plant or amonocotyledonous plant. In another embodiment, the plant is furtherselected from the group consisting of an alfalfa, banana, barley, bean,broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower,celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn,clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus,flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets,melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea,peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine,radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugar beet,sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass,tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat.

In further embodiments, seeds comprising the recombinant nucleic acidmolecules are disclosed.

In another embodiment, an insect inhibitory composition comprising therecombinant nucleic acid molecules disclosed in this application arecontemplated. The insect inhibitory composition can further comprise anucleotide sequence encoding at least one other pesticidal agent that isdifferent from said pesticidal protein. In certain embodiments, the atleast one other pesticidal agent is selected from the group consistingof an insect inhibitory protein, an insect inhibitory dsRNA molecule,and an ancillary protein. It is also contemplated that the at least oneother pesticidal agent in the insect inhibitory composition exhibitsactivity against one or more pest species of the orders Lepidoptera,Coleoptera, or Hemiptera. The at least one other pesticidal agent in theinsect inhibitory composition is in one embodiment selected from thegroup consisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B,Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras, Cry1G,Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3Avariants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35,Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400,TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901,TIC1201, TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869,TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI-AXMI-, AXMI-88, AXMI-97, AXMI-102,AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AXMI-005, AXMI134,AXMI-150, AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209,AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224zand AXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1and variants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10,DIG-657 and a DIG-11 protein.

Commodity products comprising a detectable amount of the recombinantnucleic acid molecules disclosed in this application are alsocontemplated. Such commodity products include commodity corn bagged by agrain handler, corn flakes, corn cakes, corn flour, corn meal, cornsyrup, corn oil, corn silage, corn starch, corn cereal, and the like,and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut,fruit, melon, and vegetable commodity products including, whereapplicable, juices, concentrates, jams, jellies, marmalades, and otheredible forms of such commodity products containing a detectable amountof such polynucleotides and or polypeptides of this application, wholeor processed cotton seed, cotton oil, lint, seeds and plant partsprocessed for feed or food, fiber, paper, biomasses, and fuel productssuch as fuel derived from cotton oil or pellets derived from cotton ginwaste, whole or processed soybean seed, soybean oil, soybean protein,soybean meal, soybean flour, soybean flakes, soybean bran, soybean milk,soybean cheese, soybean wine, animal feed comprising soybean, papercomprising soybean, cream comprising soybean, soybean biomass, and fuelproducts produced using soybean plants and soybean plant parts.

Also contemplated in this application is a method of producing seedcomprising the recombinant nucleic acid molecules disclosed in thisapplication. The method comprises planting at least one of the seedcomprising the recombinant nucleic acid molecules disclosed in thisapplication; growing plant from the seed; and harvesting seed from theplants, wherein the harvested seed comprises the recombinant nucleicacid molecules in this application.

In another illustrative embodiment, a plant resistant to insectinfestation, is provided wherein the cells of said plant comprise: (a) arecombinant nucleic acid molecule encoding an insecticidally effectiveamount of a pesticidal protein as set forth in SEQ ID NO:4, SEQ ID NO:2,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18; or (b) aninsecticidally effective amount of a protein comprising an amino acidsequence having at least 85%, or 90%, or 95%, or about 100% amino acidsequence identity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:16, or SEQ ID NO:18.

Also disclosed in this application are methods for controlling aLepidopteran species pest, and controlling a Lepidopteran species pestinfestation of a plant, particularly a crop plant. The method comprises,in one embodiment, (a) contacting the pest with an insecticidallyeffective amount of a pesticidal proteins as set forth in SEQ ID NO:4,SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18;or (b) contacting the pest with an insecticidally effective amount ofone or more pesticidal proteins comprising an amino acid sequence havingat least 85%, or 90%, or 95%, or about 100% amino acid sequence identityto identity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:12, SEQID NO:16, or SEQ ID NO:18.

Further provided herein is a method of detecting the presence of arecombinant nucleic acid molecule comprising a polynucleotide segmentencoding a pesticidal protein or fragment thereof, wherein: (a) saidpesticidal protein comprises the amino acid sequence of SEQ ID NO:4, SEQID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18; or (b) said pesticidal proteincomprises an amino acid sequence having at least 65%, or 70%, or 75%, or80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acidsequence identity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18;or (c) said polynucleotide segment hybridizes to a polynucleotide havingthe nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ IDNO:17. In one embodiment of the invention, the method comprisescontacting a sample of nucleic acids with a nucleic acid probe thathybridizes under stringent hybridization conditions with genomic DNAfrom a plant comprising a polynucleotide segment encoding a pesticidalprotein or fragment thereof provided herein, and does not hybridizeunder such hybridization conditions with genomic DNA from an otherwiseisogenic plant that does not comprise the segment, wherein the probe ishomologous or complementary to SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, orSEQ ID NO:17, or a sequence that encodes a pesticidal protein comprisingan amino acid sequence having at least 65%, or 70%, or 75%, or 80%, or85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequenceidentity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18. Themethod may further comprise (a) subjecting the sample and probe tostringent hybridization conditions; and (b) detecting hybridization ofthe probe with DNA of the sample.

Also provided by the invention are methods of detecting the presence ofa pesticidal protein or fragment thereof in a sample comprising protein,wherein said pesticidal protein comprises the amino acid sequence of SEQID NO:2; or said pesticidal protein comprises an amino acid sequencehaving at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO:4,SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, or SEQ ID NO:18. In one embodiment, the methodcomprises: (a) contacting a sample with an immunoreactive antibody; and(b) detecting the presence of the protein. In some embodiments the stepof detecting comprises an ELISA, or a Western blot.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleic acid sequence encoding a TIC6757 pesticidalprotein obtained from Paenibacillus popilliae species DSC004343.

SEQ ID NO:2 is the amino acid sequence of the TIC6757 pesticidalprotein.

SEQ ID NO:3 is a synthetic coding sequence encoding a TIC6757PLpesticidal protein designed for expression in a plant cell wherein anadditional alanine codon is inserted immediately following theinitiating methionine codon.

SEQ ID NO:4 is the amino acid sequence of TIC6757PL encoded by asynthetic coding sequence designed for expression in a plant cell (SEQID NO:3), and wherein an additional alanine amino acid is insertedimmediately following the initiating methionine.

SEQ ID NO:5 is a nucleic acid sequence encoding a TIC6757 His pesticidalprotein, wherein a nucleic acid sequence encoding a Histidine tag isoperably linked 5′ and in frame to the TIC6757 coding sequence.

SEQ ID NO:6 is the amino acid sequence of the TIC6757 His pesticidalprotein.

SEQ ID NO:7 is a nucleic acid sequence encoding a TIC7472 pesticidalprotein obtained from Paenibacillus popilliae species DSC007648.

SEQ ID NO:8 is the amino acid sequence of the TIC7242 pesticidalprotein.

SEQ ID NO:9 is a nucleic acid sequence encoding a TIC7472 His pesticidalprotein, wherein a nucleic acid sequence encoding a Histidine tag isoperably linked 3′ and in frame to the TIC7472 coding sequence.

SEQ ID NO:10 is the amino acid sequence of the TIC7472 His pesticidalprotein.

SEQ ID NO:11 is a nucleic acid sequence encoding a TIC7473 pesticidalprotein from an open reading frame at nucleotide position 1-2391 and atranslation termination codon.

SEQ ID NO:12 is the amino acid sequence translation of the TIC7243pesticidal protein obtained from Paenibacillus popilliae speciesDSC008493.

SEQ ID NO:13 is a recombinant nucleic acid sequence encoding a TIC7473His pesticidal protein, wherein a nucleic acid sequence encoding aHistidine tag is operably linked 3′ and in frame to the TIC7472 codingsequence.

SEQ ID NO:14 is the amino acid sequence translation of the TIC7473 Hispesticidal protein.

SEQ ID NO:15 is a synthetic coding sequence encoding a TIC7472PLpesticidal protein designed for expression in a plant cell wherein anadditional alanine codon is inserted immediately following theinitiating methionine codon.

SEQ ID NO:16 is the amino acid sequence of TIC7472PL encoded by asynthetic coding sequence designed for expression in a plant cell (SEQID NO:15), and wherein an additional alanine amino acid is insertedimmediately following the initiating methionine.

SEQ ID NO:17 is a synthetic coding sequence encoding a TIC7473PLpesticidal protein designed for expression in a plant cell wherein anadditional alanine codon is inserted immediately following theinitiating methionine codon.

SEQ ID NO:18 is the amino acid sequence of TIC7473PL encoded by asynthetic coding sequence designed for expression in a plant cell (SEQID NO:17), and wherein an additional alanine amino acid is insertedimmediately following the initiating methionine.

DETAILED DESCRIPTION OF THE INVENTION

The problem in the art of agricultural pest control can be characterizedas a need for new toxin proteins that are efficacious against targetpests, exhibit broad spectrum toxicity against target pest species, arecapable of being expressed in plants without causing undesirableagronomic issues, and provide an alternative mode of action compared tocurrent toxins that are used commercially in plants.

Novel pesticidal proteins exemplified by TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, and TIC7473PL are disclosed herein, and address eachof these needs, particularly against a broad spectrum of Lepidopteraninsect pests, and more particularly against Black armyworm (Spodopteraexempta), Black cutworm (Agrotis ipsilon), Corn earworm (Helicoverpazea), Cotton leaf worm (Alabama argillacea), Diamondback moth (Plutellaxylostella), European corn borer (Ostrinia nubilalis), Fall armyworm(Spodoptera frugiperda), Cry1Fa1 resistant Fall armyworm (Spodopterafrugiperda), Old World bollworm (OWB, Helicoverpa armigera), Southernarmyworm (Spodoptera eridania), Soybean looper (Chrysodeixis includens),Spotted bollworm (Earias vittella), Southwestern corn borer (Diatraeagrandiosella), Tobacco budworm (Heliothis virescens), Tobacco cutworm(Spodoptera litura, also known as cluster caterpillar), Western beancutworm (Striacosta albicosta), and Velvet bean caterpillar (Anticarsiagemmatalis).

Reference in this application to TIC6757, “TIC6757 protein”, “TIC6757protein toxin”, “TIC6757 toxin protein”, “TIC6757 pesticidal protein”,“TIC6757-related toxins”, “TIC6757-related toxin proteins”, TIC6757PL,“TIC6757PL protein”, “TIC6757PL protein toxin”, “TIC6757PL toxinprotein”, “TIC6757PL pesticidal protein”, “TIC6757PL-related toxins”,“TIC6757PL-related toxin proteins”, TIC7472, “TIC7472 protein”, “TIC7472protein toxin”, “TIC7472 toxin protein”, “TIC7472 pesticidal protein”,“TIC7472-related toxins”, “TIC7472-related toxin proteins”, TIC7472PL,“TIC7472PL protein”, “TIC7472PL protein toxin”, “TIC7472PL toxinprotein”, “TIC7472PL pesticidal protein”, “TIC7472PL-related toxins”,“TIC7472PL-related toxin proteins”, TIC7473, “TIC7473 protein”, “TIC7473protein toxin”, “TIC7473 toxin protein”, “TIC7473 pesticidal protein”,“TIC7473-related toxins”, “TIC7473-related toxin proteins”, TIC7473PL,“TIC7473PL protein”, “TIC7473PL protein toxin”, “TIC7473PL toxinprotein”, “TIC7473PL pesticidal protein”, “TIC7473PL-related toxins”,“TIC7473PL-related toxin proteins”, and the like, refer to any novelpesticidal protein or insect inhibitory protein, that comprises, thatconsists of, that is substantially homologous to, that is similar to, orthat is derived from any pesticidal protein or insect inhibitory proteinsequence of TIC6757 (SEQ ID NO:2), TIC6757PL (SEQ ID NO:4), TIC7472 (SEQID NO:8). TIC7472PL (SEQ ID NO:16), TIC7473 (SEQ ID NO:12), or TIC7473PL(SEQ ID NO:18) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Lepidopteran pests,including any protein exhibiting pesticidal or insect inhibitoryactivity if alignment of such protein with TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL results in amino acid sequence identityof any fraction percentage form about 85% to about 100% percent. TheTIC6757 and TIC6757PL proteins include both the plastid-targeted andnon-plastid targeted form of the proteins.

The term “segment” or “fragment” is used in this application to describeconsecutive amino acid or nucleic acid sequences that are shorter thanthe complete amino acid or nucleic acid sequence describing a TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL protein. A segmentor fragment exhibiting insect inhibitory activity is also disclosed inthis application if alignment of such segment or fragment, with thecorresponding section of the TIC6757 protein set forth in SEQ ID NO:2,TIC6757PL protein set forth in SEQ ID NO:4, TIC7472 protein set forth inSEQ ID NO:8, TIC7472PL protein set forth in SEQ ID NO:16, TIC7473protein set forth in SEQ ID NO:12, or TIC7473PL protein set forth in SEQID NO:18, results in amino acid sequence identity of any fractionpercentage from about 85 to about 100 percent between the segment orfragment and the corresponding section of the TIC6757, TIC6757PL,TIC7472, TIC7472PL, TIC7473, or TIC7473PL protein.

Reference in this application to the terms “active” or “activity”,“pesticidal activity” or “pesticidal” or “insecticidal activity”,“insect inhibitory” or “insecticidal” refer to efficacy of a toxicagent, such as a protein toxin, in inhibiting (inhibiting growth,feeding, fecundity, or viability), suppressing (suppressing growth,feeding, fecundity, or viability), controlling (controlling the pestinfestation, controlling the pest feeding activities on a particularcrop containing an effective amount of the TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL protein) or killing (causing themorbidity, mortality, or reduced fecundity of) a pest. These terms areintended to include the result of providing a pesticidally effectiveamount of a toxic protein to a pest where the exposure of the pest tothe toxic protein results in morbidity, mortality, reduced fecundity, orstunting. These terms also include repulsion of the pest from the plant,a tissue of the plant, a plant part, seed, plant cells, or from theparticular geographic location where the plant may be growing, as aresult of providing a pesticidally effective amount of the toxic proteinin or on the plant. In general, pesticidal activity refers to theability of a toxic protein to be effective in inhibiting the growth,development, viability, feeding behavior, mating behavior, fecundity, orany measurable decrease in the adverse effects caused by an insectfeeding on this protein, protein fragment, protein segment orpolynucleotide of a particular target pest, including but not limited toinsects of the order Lepidoptera. The toxic protein can be produced bythe plant or can be applied to the plant or to the environment withinthe location where the plant is located. The terms “bioactivity”,“effective”, “efficacious” or variations thereof are also termsinterchangeably utilized in this application to describe the effects ofproteins of the present invention on target insect pests.

A pesticidally effective amount of a toxic agent, when provided in thediet of a target pest, exhibits pesticidal activity when the toxic agentcontacts the pest. A toxic agent can be a pesticidal protein or one ormore chemical agents known in the art. Pesticidal or insecticidalchemical agents and pesticidal or insecticidal protein agents can beused alone or in combinations with each other. Chemical agents includebut are not limited to dsRNA molecules targeting specific genes forsuppression in a target pest, organochlorides, organophosphates,carbamates, pyrethroids, neonicotinoids, and ryanoids. Pesticidal orinsecticidal protein agents include the protein toxins set forth in thisapplication, as well as other proteinaceous toxic agents including thosethat target Lepidopterans, as well as protein toxins that are used tocontrol other plant pests such as Cry and Cyt proteins available in theart for use in controlling Coleopteran, Hemipteran and Homopteranspecies.

It is intended that reference to a pest, particularly a pest of a cropplant, means insect pests of crop plants, particularly those Lepidopterainsect pests that are controlled by the TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL protein toxin class. However, referenceto a pest can also include Coleopteran, Hemipteran and Homopteran insectpests of plants, as well as nematodes and fungi when toxic agentstargeting these pests are co-localized or present together with theTIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL protein ora protein that is 85 to about 100 percent identical to TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL.

The TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, and TIC7473PLproteins are related by a common function and exhibit insecticidalactivity towards insect pests from the Lepidoptera insect species,including adults, pupae, larvae, and neonates.

The insects of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the Family Noctuidae,e.g., Fall armyworm (Spodoptera frugiperda), Beet armyworm (Spodopteraexigua), Black armyworm (Spodoptera exempta), Southern armyworm(Spodoptera eridania), bertha armyworm (Mamestra configurata), blackcutworm (Agrotis ipsilon), cabbage looper (Trichoplusia ni), soybeanlooper (Pseudoplusia includens), velvetbean caterpillar (Anticarsiagemmatalis), green cloverworm (Hypena scabra), tobacco budworm(Heliothis virescens), granulate cutworm (Agrotis subterranea), armyworm(Pseudaletia unipuncta), western cutworm (Agrotis orthogonia); borers,casebearers, webworms, coneworms, cabbageworms and skeletonizers fromthe Family Pyralidae, e.g., European corn borer (Ostrinia nubilalis),navel orangeworm (Amyelois transitella), corn root webworm (Crambuscaliginosellus), sod webworm (Herpetogramma licarsisalis), sunflowermoth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpuslignosellus); leafrollers, budworms, seed worms, and fruit worms in theFamily Tortricidae, e.g., codling moth (Cydia pomonella), grape berrymoth (Endopiza viteana), oriental fruit moth (Grapholita molesta),sunflower bud moth (Suleima helianthana); and many other economicallyimportant Lepidoptera, e.g., diamondback moth (Plutella xylostella),pink bollworm (Pectinophora gossypiella), and gypsy moth (Lymantriadispar). Other insect pests of order Lepidoptera include, e.g., cottonleaf worm (Alabama argillacea), fruit tree leaf roller (Archipsargyrospila), European leafroller (Archips rosana) and other Archipsspecies, (Chilo suppressalis, Asiatic rice borer, or rice stem borer),rice leaf roller (Cnaphalocrocis medinalis), corn root webworm (Crambuscaliginosellus), bluegrass webworm (Crambus teterrellus), southwesterncorn borer (Diatraea grandiosella), surgarcane borer (Diatraeasaccharalis), spiny bollworm (Earias insulana), spotted bollworm (Eariasvittella), American bollworm (Helicoverpa armigera), corn earworm(Helicoverpa zea, also known as soybean podworm and cotton bollworm),tobacco budworm (Heliothis virescens), sod webworm (Herpetogrammalicarsisalis), Western bean cutworm (Striacosta albicosta), Europeangrape vine moth (Lobesia botrana), citrus leafminer (Phyllocnistiscitrella), large white butterfly (Pieris brassicae), small whitebutterfly (Pieris rapae, also known as imported cabbageworm), beetarmyworm (Spodoptera exigua), tobacco cutworm (Spodoptera litura, alsoknown as cluster caterpillar), and tomato leafminer (Tuta absoluta).

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

As described further in this application, an open reading frame (ORF)encoding TIC6757 (SEQ ID NO:19) was discovered in DNA obtained fromPaenibacillus popilliae strain DSC004343. The coding sequence was clonedand expressed in microbial host cells to produce recombinant proteinsused in bioassays. High throughput screening and bioinformaticstechniques were used to screen microbial sequences for genes encodingproteins exhibiting similarity to TIC6757. An open reading frame (ORF)encoding TIC7472 (SEQ ID NO:7) was discovered in DNA obtained fromPaenibacillus popilliae strain DSC007648. An open reading frame (ORF)encoding TIC7473 (SEQ ID NO:11) was discovered in DNA obtained fromPaenibacillus popilliae strain DSC008493. Bioassay using microbial hostcell-derived proteins of TIC6757 demonstrated activity against theLepidopteran species Beet armyworm (Spodoptera exigua), Black cutworm(Agrotis ipsilon), Corn earworm (Helicoverpa zea), Cotton leaf worm(Alabama argillacea), Diamondback moth (Plutella xylostella), Europeancorn borer (Ostrinia nubilalis), Fall armyworm (Spodoptera frugiperda),Cry1Fa1 resistant Fall armyworm (Spodoptera frugiperda), Old Worldbollworm (OWB, Helicoverpa armigera), Southern armyworm (Spodopteraeridania), Soybean looper (Chrysodeixis includens), Spotted bollworm(Earias vittella), Southwestern corn borer (Diatraea grandiosella),Tobacco budworm (Heliothis virescens), Tobacco cutworm (Spodopteralitura, also known as cluster caterpillar), and Velvet bean caterpillar(Anticarsia gemmatalis). Bioassay using microbial host cell-derivedproteins of TIC7472 and TIC7473 demonstrated activity against theLepidopteran species Corn earworm (Helicoverpa zea), Fall armyworm(Spodoptera frugiperda), Southern armyworm (Spodoptera eridania),Soybean looper (Chrysodeixis includens), and Southwestern corn borer(Diatraea grandiosella).

For expression in plant cells, the TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, and TIC7473PL proteins can be expressed to reside inthe cytosol or targeted to various organelles of the plant cell. Forexample, targeting a protein to the chloroplast may result in increasedlevels of expressed protein in a transgenic plant while preventingoff-phenotypes from occurring. Targeting may also result in an increasein pest resistance efficacy in the transgenic event. A target peptide ortransit peptide is a short (3-70 amino acids long) peptide chain thatdirects the transport of a protein to a specific region in the cell,including the nucleus, mitochondria, endoplasmic reticulum (ER),chloroplast, apoplast, peroxisome and plasma membrane. Some targetpeptides are cleaved from the protein by signal peptidases after theproteins are transported. For targeting to the chloroplast, proteinscontain transit peptides which are around 40-50 amino acids. Fordescriptions of the use of chloroplast transit peptides, see U.S. Pat.Nos. 5,188,642 and 5,728,925. Many chloroplast-localized proteins areexpressed from nuclear genes as precursors and are targeted to thechloroplast by a chloroplast transit peptide (CTP). Examples of suchisolated chloroplast proteins include, but are not limited to, thoseassociated with the small subunit (SSU) of ribulose-1,5,-bisphosphatecarboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvestingcomplex protein I and protein II, thioredoxin F, enolpyruvyl shikimatephosphate synthase (EPSPS), and transit peptides described in U.S. Pat.No. 7,193,133. It has been demonstrated in vivo and in vitro thatnon-chloroplast proteins may be targeted to the chloroplast by use ofprotein fusions with a heterologous CTP and that the CTP is sufficientto target a protein to the chloroplast. Incorporation of a suitablechloroplast transit peptide such as the Arabidopsis thaliana EPSPS CTP(CTP2) (see, Klee et al., Mol. Gen. Genet. 210:437-442, 1987) or thePetunia hybrida EPSPS CTP (CTP4) (see, della-Cioppa et al., Proc. Natl.Acad. Sci. USA 83:6873-6877, 1986) has been shown to target heterologousEPSPS protein sequences to chloroplasts in transgenic plants (see, U.S.Pat. Nos. 5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP189707; EP 508909; and EP 924299). For targeting the TIC6757 orTIC6757PL toxin protein to the chloroplast, a sequence encoding achloroplast transit peptide is placed 5′ in operable linkage and inframe to a synthetic coding sequence encoding the TIC6757 or TIC6757PLtoxin protein that has been designed for optimal expression in plantcells.

It is contemplated that additional toxin protein sequences related toTIC6757, TIC7472, and TIC7473 can be created by using the amino acidsequence of TIC6757, TIC7472, or TIC7473 to create novel proteins withnovel properties. The TIC6757, TIC7472, and TIC7473 toxin proteins canbe aligned to combine differences at the amino acid sequence level intonovel amino acid sequence variants and making appropriate changes to therecombinant nucleic acid sequence encoding the variants.

This disclosure further contemplates that improved variants of theTIC6757 protein toxin class can be engineered in planta by using variousgene editing methods known in the art. Such technologies used for genomeediting include, but are not limited to, ZFN (zinc-finger nuclease),meganucleases, TALEN (Transcription activator-like effector nucleases),and CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR-associated) systems. These genome editing methodscan be used to alter the toxin protein coding sequence transformedwithin a plant cell to a different toxin coding sequence. Specifically,through these methods, one or more codons within the toxin codingsequence is altered to engineer a new protein amino acid sequence.Alternatively, a fragment within the coding sequence is replaced ordeleted, or additional DNA fragments are inserted into the codingsequence, to engineer a new toxin coding sequence. The new codingsequence can encode a toxin protein with new properties such asincreased activity or spectrum against insect pests, as well as provideactivity against an insect pest species wherein resistance has developedagainst the original insect toxin protein. The plant cell comprising thegene edited toxin coding sequence can be used by methods known in theart to generate whole plants expressing the new toxin protein.

It is also contemplated that fragments of TIC6757, TIC7472, and TIC7473or protein variants thereof can be truncated forms wherein one or moreamino acids are deleted from the N-terminal end, C-terminal end, themiddle of the protein, or combinations thereof wherein the fragments andvariants retain insect inhibitory activity. These fragments can benaturally occurring or synthetic variants of TIC6757, TIC7472, andTIC7473 or derived protein variants, but should retain the insectinhibitory activity of at least TIC6757, TIC7472, or TIC7473.

Proteins that resemble the TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, and TIC7473PL proteins can be identified and compared to eachother using various computer based algorithms known in the art (seeTables 1 and 2). Amino acid sequence identities reported in thisapplication are a result of a Clustal W alignment using these defaultparameters: Weight matrix: blosum, Gap opening penalty: 10.0, Gapextension penalty: 0.05, Hydrophilic gaps: On, Hydrophilic residues:GPSNDQERK, Residue-specific gap penalties: On (Thompson, et al (1994)Nucleic Acids Research, 22:4673-4680). Percent amino acid identity isfurther calculated by the product of 100% multiplied by (amino acididentities/length of subject protein). Other alignment algorithms arealso available in the art and provide results similar to those obtainedusing a Clustal W alignment and are contemplated herein.

It is intended that a protein exhibiting insect inhibitory activityagainst a Lepidopteran insect species is related to TIC6757, TIC6757PL,TIC7472, TIC7472PL, TIC7473, or TIC7473PL if the protein is used in aquery, e.g., in a Clustal W alignment, and the proteins of the presentinvention as set forth as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ IDNO:16, SEQ ID NO:12, or SEQ ID NO:18 are identified as hits in suchalignment in which the query protein exhibits at least 85% to about 100%amino acid identity along the length of the query protein that is about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100%, or any fraction percentage in this range.

Exemplary proteins TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, andTIC7473PL were aligned with each other using a Clustal W algorithm. Apair-wise matrix of percent amino acid sequence identities for each ofthe full-length proteins was created, as reported in Table 1.

TABLE 1 Pair-wise matrix display of exemplary proteins TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, and TIC7473PL. TIC6757 TIC6757PLTIC7472 TIC7472PL TIC7473 TIC7473PL Toxin (SEQ ID NO: 2) (SEQ ID NO: 4)(SEQ ID NO: 8) (SEQ ID NO: 16) (SEQ ID NO: 12) (SEQ ID NO: 18) TIC6757 —99.9 (796) 99.7 (795) 99.6 (794) 99.9 (796) 99.7 (795) (SEQ ID NO: 2)TIC6757PL 99.7 (796) — 99.5 (794) 99.7 (796) 99.6 (795) 99.9 (797) (SEQID NO: 4) TIC7472 99.7 (795) 99.6 (794) — 99.9 (796) 99.9 (796) 99.7(795) (SEQ ID NO: 8) TIC7472PL 99.5 (794) 99.7 (796) 99.7 (796) — 99.6(795) 99.9 (797) (SEQ ID NO: 16) TIC7473 99.9 (796) 99.7 (795) 99.9(796) 99.7 (795) — 99.9 (796) (SEQ ID NO: 12) TIC7473PL 99.6 (795) 99.9(797) 99.6 (795) 99.9 (797) 99.7 (796) — (SEQ ID NO: 18) TableDescription: Clustal W alignment between (X) and (Y) are reported in apair-wise matrix. The percent amino acid identity between all pairs iscalculated and is represented by the first number in each box. Thesecond number (in parentheses) in each box represents the number ofidentical amino acids between the pair.

In addition to percent identity, TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, TIC7473PL and related proteins can also be related by primarystructure (conserved amino acid motifs), by length (about 797 aminoacids), and by other characteristics. Characteristics of the TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, and TIC7473PL protein toxins arereported in Table 2.

TABLE 2 Selected characteristics of the TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, and TIC7473PL proteins. Molecular Amino No. of No.of No. of No. of Weight Acid Isoelectric Charge Strongly Basic (−)Strongly Acidic Hydrophobic Polar Protein (in Daltons) Length Point atPH 7.0 Amino Acids Amino Acids Amino Acids Amino Acids TIC6757 90011.21797 4.4289 −34.5 81 112 391 406 TIC6757PL 90082.29 798 4.4289 −34.5 81112 392 406 TIC7472 90096.28 797 4.4141 −35.5 81 113 390 407 TIC7472PL90167.36 798 4.4141 −35.5 81 113 391 407 TIC7473 90069.25 797 4.4141−35.5 81 113 390 407 TIC7473PL 90140.33 798 4.4141 −35.5 81 113 391 407

As described further in the Examples of this application, a syntheticnucleic acid molecule sequence encoding a variant of TIC6757, TIC6757PLwas designed for use in plants. An exemplary recombinant nucleic acidmolecule sequence that was designed for use in plants encoding theTIC6757PL protein is presented as SEQ ID NO:3. The TIC6757PL protein hasan additional alanine amino acid immediately following the initiatingmethionine relative to the TIC6757 protein. The additional alanineresidue inserted into the TIC6757 amino acid sequence is believed toimprove expression of the protein in planta. Likewise, synthetic nucleicacid molecule sequences encoding variants of TIC7472 and TIC7473 arereferred to herein as TIC7472PL and TIC7473PL, respectively, and weredesigned for use in plants. Exemplary synthetic nucleic acid moleculesequences that were designed for use in plants encoding TIC7472PL andTIC7473PL are presented as SEQ ID NO:15 and SEQ ID NO:17, respectively.Both the TIC7472PL and TIC7473PL proteins have an additional alanineamino acid immediately following the initiating methionine relative tothe TIC7472 and TIC7473 proteins.

Expression cassettes and vectors containing a recombinant nucleic acidmolecule sequence can be constructed and introduced into corn, soybeanor cotton plant cells in accordance with transformation methods andtechniques known in the art. For example, Agrobacterium-mediatedtransformation is described in U.S. Patent Application Publications2009/0138985A1 (soybean), 2008/0280361A1 (soybean), 2009/0142837A1(corn), 2008/0282432 (cotton), 2008/0256667 (cotton), 2003/0110531(wheat), 2001/0042257 A1 (sugar beet), U.S. Pat. No. 5,750,871 (canola),U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat. No. 6,365,807 (rice), andin Arencibia et al. (1998) Transgenic Res. 7:213-222 (sugarcane) all ofwhich are incorporated herein by reference in their entirety.Transformed cells can be regenerated into transformed plants thatexpress TIC6757PL, TIC7472 and TIC7473 proteins and demonstratepesticidal activity through bioassays performed in the presence ofLepidopteran pest larvae using plant leaf disks obtained from thetransformed plants. Plants can be derived from the plant cells byregeneration, seed, pollen, or meristem transformation techniques.Methods for transforming plants are known in the art.

As an alternative to traditional transformation methods, a DNA sequence,such as a transgene, expression cassette(s), etc., may be inserted orintegrated into a specific site or locus within the genome of a plant orplant cell via site-directed integration. Recombinant DNA construct(s)and molecule(s) of this disclosure may thus include a donor templatesequence comprising at least one transgene, expression cassette, orother DNA sequence for insertion into the genome of the plant or plantcell. Such donor template for site-directed integration may furtherinclude one or two homology arms flanking an insertion sequence (i.e.,the sequence, transgene, cassette, etc., to be inserted into the plantgenome). The recombinant DNA construct(s) of this disclosure may furthercomprise an expression cassette(s) encoding a site-specific nucleaseand/or any associated protein(s) to carry out site-directed integration.These nuclease expressing cassette(s) may be present in the samemolecule or vector as the donor template (in cis) or on a separatemolecule or vector (in trans). Several methods for site-directedintegration are known in the art involving different proteins (orcomplexes of proteins and/or guide RNA) that cut the genomic DNA toproduce a double strand break (DSB) or nick at a desired genomic site orlocus. Briefly as understood in the art, during the process of repairingthe DSB or nick introduced by the nuclease enzyme, the donor templateDNA may become integrated into the genome at the site of the DSB ornick. The presence of the homology arm(s) in the donor template maypromote the adoption and targeting of the insertion sequence into theplant genome during the repair process through homologous recombination,although an insertion event may occur through non-homologous end joining(NHEJ). Examples of site-specific nucleases that may be used includezinc-finger nucleases, engineered or native meganucleases,TALE-endonucleases, and RNA-guided endonucleases (e.g., Cas9 or Cpf1).For methods using RNA-guided site-specific nucleases (e.g., Cas9 orCpf1), the recombinant DNA construct(s) will also comprise a sequenceencoding one or more guide RNAs to direct the nuclease to the desiredsite within the plant genome.

Recombinant nucleic acid molecule compositions that encode TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, and TIC7473PL are contemplated.For example, TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, andTIC7473PL proteins can be expressed with recombinant DNA constructs inwhich a polynucleotide molecule with an ORF encoding the protein isoperably linked to genetic expression elements such as a promoter andany other regulatory element necessary for expression in the system forwhich the construct is intended. Non-limiting examples include aplant-functional promoter operably linked to a TIC6757PL, TIC7472PL, orTIC7473PL protein encoding sequence for expression of the protein inplants or a Bt-functional promoter operably linked to a TIC6757,TIC7472, or TIC7473 protein encoding sequence for expression of theprotein in a Bt bacterium or other Bacillus species. Other elements canbe operably linked to the TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, or TIC7473PL protein encoding sequence including, but notlimited to, enhancers, introns, untranslated leaders, encoded proteinimmobilization tags (HIS-tag), translocation peptides (i.e., plastidtransit peptides, signal peptides), polypeptide sequences forpost-translational modifying enzymes, ribosomal binding sites, and RNAitarget sites. Exemplary recombinant polynucleotide molecules providedherewith include, but are not limited to, a heterologous promoteroperably linked to a polynucleotide such as SEQ ID NO:3, SEQ ID NO:1,SIQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:17 that encodesthe respective polypeptides or proteins having the amino acid sequenceas set forth in SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:12, SEQID NO:16, and SEQ ID NO:18. A heterologous promoter can also be operablylinked to synthetic DNA coding sequences encoding a plastid targetedTIC6757PL, TIC7472PL, or TIC7473PL; or an untargeted TIC6757PL,TIC7472PL, or TIC7473PL. The codons of a recombinant nucleic acidmolecule encoding for proteins disclosed herein can be substituted bysynonymous codons (known in the art as a silent substitution).

A recombinant DNA construct comprising TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL protein encoding sequences can furthercomprise a region of DNA that encodes for one or more insect inhibitoryagents which can be configured to concomitantly express or co-expresswith a DNA sequence encoding a TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, or TIC7473PL protein, a protein different from a TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL protein, an insectinhibitory dsRNA molecule, or an ancillary protein. Ancillary proteinsinclude, but are not limited to, co-factors, enzymes, binding-partners,or other agents that function to aid in the effectiveness of an insectinhibitory agent, for example, by aiding its expression, influencing itsstability in plants, optimizing free energy for oligomerization,augmenting its toxicity, and increasing its spectrum of activity. Anancillary protein may facilitate the uptake of one or more insectinhibitory agents, for example, or potentiate the toxic effects of thetoxic agent.

A recombinant DNA construct can be assembled so that all proteins ordsRNA molecules are expressed from one promoter or each protein or dsRNAmolecules is under separate promoter control or some combinationthereof. The proteins of this invention can be expressed from amulti-gene expression system in which one or more proteins of TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL are expressed froma common nucleotide segment which also contains other open readingframes and promoters, depending on the type of expression systemselected. For example, a bacterial multi-gene expression system canutilize a single promoter to drive expression of multiply-linked/tandemopen reading frames from within a single operon (i.e., polycistronicexpression). In another example, a plant multi-gene expression systemcan utilize multiply-unlinked or linked expression cassettes, eachcassette expressing a different protein or other agent such as one ormore dsRNA molecules.

Recombinant polynucleotides or recombinant DNA constructs comprising aTIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL proteinencoding sequence can be delivered to host cells by vectors, e.g., aplasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid,phage, or viral vector. Such vectors can be used to achieve stable ortransient expression of a TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, or TIC7473PL protein encoding sequence in a host cell, orsubsequent expression of the encoded polypeptide. An exogenousrecombinant polynucleotide or recombinant DNA construct that comprises aTIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL proteinencoding sequence and that is introduced into a host cell is referred inthis application as a “transgene”.

Transgenic bacteria, transgenic plant cells, transgenic plants, andtransgenic plant parts that contain a recombinant polynucleotide thatexpresses any one or more of TIC6757 or a related family toxin proteinencoding sequence are provided herein. The term “bacterial cell” or“bacterium” can include, but is not limited to, an Agrobacterium, aBacillus, an Escherichia, a Salmonella, a Pseudomonas, Brevibacillus,Klebsiella, Erwinia, or a Rhizobium cell. The term “plant cell” or“plant” can include but is not limited to a dicotyledonous ormonocotyledonous plant. The term “plant cell” or “plant” can alsoinclude but is not limited to an alfalfa, banana, barley, bean,broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower,celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn,clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus,flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets,melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea,peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine,radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugar beet,sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass,tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plantcell or plant. In certain embodiments, transgenic plants and transgenicplant parts regenerated from a transgenic plant cell are provided. Incertain embodiments, the transgenic plants can be obtained from atransgenic seed, by cutting, snapping, grinding or otherwisedisassociating the part from the plant. In certain embodiments, theplant part can be a seed, a boll, a leaf, a flower, a stem, a root, orany portion thereof, or a non-regenerable portion of a transgenic plantpart. As used in this context, a “non-regenerable” portion of atransgenic plant part is a portion that can not be induced to form awhole plant or that can not be induced to form a whole plant that iscapable of sexual and/or asexual reproduction. In certain embodiments, anon-regenerable portion of a plant part is a portion of a transgenicseed, boll, leaf, flower, stem, or root.

Methods of making transgenic plants that comprise insect,Lepidoptera-inhibitory amounts of a TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL protein are provided. Such plants canbe made by introducing a recombinant polynucleotide that encodes any ofthe proteins provided in this application into a plant cell, andselecting a plant derived from said plant cell that expresses an insect,Lepidoptera-inhibitory amount of the proteins. Plants can be derivedfrom the plant cells by regeneration, seed, pollen, or meristemtransformation techniques. Methods for transforming plants are known inthe art.

Processed plant products, wherein the processed product comprises adetectable amount of a TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473,or TIC7473PL protein, an insect inhibitory segment or fragment thereof,or any distinguishing portion thereof, are also disclosed herein. Incertain embodiments, the processed product is selected from the groupconsisting of plant parts, plant biomass, oil, meal, sugar, animal feed,flour, flakes, bran, lint, hulls, processed seed, and seed. In certainembodiments, the processed product is non-regenerable. The plant productcan comprise commodity or other products of commerce derived from atransgenic plant or transgenic plant part, where the commodity or otherproducts can be tracked through commerce by detecting nucleotidesegments or expressed RNA or proteins that encode or comprisedistinguishing portions of a TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, or TIC7473PL protein.

Plants expressing the TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473,or TIC7473PL proteins can be crossed by breeding with transgenic eventsexpressing other toxin proteins and/or expressing other transgenictraits such as herbicide tolerance genes, genes conferring yield orstress tolerance traits, and the like, or such traits can be combined ina single vector so that the traits are all linked.

As further described in the Examples, TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL protein-encoding sequences andsequences having a substantial percentage identity to TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL can be identifiedusing methods known to those of ordinary skill in the art such aspolymerase chain reaction (PCR), thermal amplification andhybridization. For example, the proteins TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL can be used to produce antibodies thatbind specifically to related proteins, and can be used to screen for andto find other protein members that are closely related.

Furthermore, nucleotide sequences encoding the TIC6757, TIC6757PL,TIC7472, TIC7472PL, TIC7473, and TIC7473PL toxin proteins can be used asprobes and primers for screening to identify other members of the classusing thermal-cycle or isothermal amplification and hybridizationmethods. For example, oligonucleotides derived from sequence as setforth in SEQ ID NO:3, SEQ ID NO:15, or SEQ ID NO:17 can be used todetermine the presence or absence of a TIC6757PL, TIC7472PL, orTIC7473PL transgene in a deoxyribonucleic acid sample derived from acommodity product. Given the sensitivity of certain nucleic aciddetection methods that employ oligonucleotides, it is anticipated thatoligonucleotides derived from sequences as set forth in SEQ ID NO:3, SEQID NO:15, and SEQ ID NO:17 can be used to detect a TIC6757PL, TIC7472PL,and TIC7473PL transgene in commodity products derived from pooledsources where only a fraction of the commodity product is derived from atransgenic plant containing any of the transgenes. It is furtherrecognized that such oligonucleotides can be used to introducenucleotide sequence variation in each of SEQ ID NO:3, SEQ ID NO:15, andSEQ ID NO:17. Such “mutagenesis” oligonucleotides are useful foridentification of TIC6757PL, TIC7472PL, and TIC7473PL amino acidsequence variants exhibiting a range of insect inhibitory activity orvaried expression in transgenic plant host cells.

Nucleotide sequence homologs, e.g., insecticidal proteins encoded bynucleotide sequences that hybridize to each or any of the sequencesdisclosed in this application under stringent hybridization conditions,are also an embodiment of the present invention. The invention alsoprovides a method for detecting a first nucleotide sequence thathybridizes to a second nucleotide sequence, wherein the first nucleotidesequence (or its reverse complement sequence) encodes a pesticidalprotein or pesticidal fragment thereof and hybridizes to the secondnucleotide sequence. In such case, the second nucleotide sequence can beany of the nucleotide sequences presented as SEQ ID NO:3, SEQ ID NO:1,SIQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, or SEQ ID NO:17 under stringenthybridization conditions. Nucleotide coding sequences hybridize to oneanother under appropriate hybridization conditions, such as stringenthybridization conditions, and the proteins encoded by these nucleotidesequences cross react with antiserum raised against any one of the otherproteins. Stringent hybridization conditions, as defined herein,comprise at least hybridization at 42° C. followed by two washes forfive minutes each at room temperature with 2×SSC, 0.1% SDS, followed bytwo washes for thirty minutes each at 65° C. in 0.5×SSC, 0.1% SDS.Washes at even higher temperatures constitute even more stringentconditions, e.g., hybridization conditions of 68° C., followed bywashing at 68° C., in 2×SSC containing 0.1% SDS.

One skilled in the art will recognize that, due to the redundancy of thegenetic code, many other sequences are capable of encoding such relatedproteins, and those sequences, to the extent that they function toexpress pesticidal proteins either in Bacillus strains or in plantcells, are embodiments of the present invention, recognizing of coursethat many such redundant coding sequences will not hybridize under theseconditions to the native Bacillus or Paenibacillus sequences encodingTIC6757, TIC7472, and TIC7473. This application contemplates the use ofthese and other identification methods known to those of ordinary skillin the art, to identify TIC6757, TIC7472, and TIC7473 protein-encodingsequences and sequences having a substantial percentage identity toTIC6757, TIC7472, and TIC7473 protein-encoding sequences.

This disclosure also contemplates the use of molecular methods known inthe art to engineer and clone commercially useful proteins comprisingchimeras of proteins from pesticidal proteins; e.g., the chimeras may beassembled from segments of the TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, or TIC7473PL proteins to derive additional useful embodimentsincluding assembly of segments of TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL proteins with segments of diverseproteins different from TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473,or TIC7473PL and related proteins. The TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL proteins may be subjected to alignmentto each other and to other Bacillus, Paenibacillus or other pesticidalproteins (whether or not these are closely or distantly relatedphylogenetically), and segments of each such protein may be identifiedthat are useful for substitution between the aligned proteins, resultingin the construction of chimeric proteins. Such chimeric proteins can besubjected to pest bioassay analysis and characterized for the presenceor absence of increased bioactivity or expanded target pest spectrumcompared to the parent proteins from which each such segment in thechimera was derived. The pesticidal activity of the polypeptides may befurther engineered for activity to a particular pest or to a broaderspectrum of pests by swapping domains or segments with other proteins orby using directed evolution methods known in the art.

Methods of controlling insects, in particular Lepidoptera infestationsof crop plants, with the TIC6757, TIC6757PL, TIC7472, TIC7472PL,TIC7473, or TIC7473PL proteins are also disclosed in this application.Such methods can comprise growing a plant comprising an insect- orLepidoptera-inhibitory amount of a TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL toxin protein. In certain embodiments,such methods can further comprise any one or more of: (i) applying anycomposition comprising or encoding a TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL toxin protein to a plant or a seed thatgives rise to a plant; and (ii) transforming a plant or a plant cellthat gives rise to a plant with a polynucleotide encoding a TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL toxin protein. Ingeneral, it is contemplated that a TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL toxin protein can be provided in acomposition, provided in a microorganism, or provided in a transgenicplant to confer insect inhibitory activity against Lepidopteran insects.

In certain embodiments, a recombinant nucleic acid molecule of TIC6757,TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL toxin proteins isthe insecticidally active ingredient of an insect inhibitory compositionprepared by culturing recombinant Bacillus or any other recombinantbacterial cell transformed to express a TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL toxin protein under conditions suitableto express the TIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, orTIC7473PL toxin protein. Such a composition can be prepared bydesiccation, lyophilization, homogenization, extraction, filtration,centrifugation, sedimentation, or concentration of a culture of suchrecombinant cells expressing/producing said recombinant polypeptide.Such a process can result in a Bacillus or other entomopathogenicbacterial cell extract, cell suspension, cell homogenate, cell lysate,cell supernatant, cell filtrate, or cell pellet. By obtaining therecombinant polypeptides so produced, a composition that includes therecombinant polypeptides can include bacterial cells, bacterial spores,and parasporal inclusion bodies and can be formulated for various uses,including as agricultural insect inhibitory spray products or as insectinhibitory formulations in diet bioassays.

In one embodiment, to reduce the likelihood of resistance development,an insect inhibitory composition comprising TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL can further comprise at least oneadditional polypeptide that exhibits insect inhibitory activity againstthe same Lepidopteran insect species, but which is different from theTIC6757, TIC6757PL, TIC7472, TIC7472PL, TIC7473, or TIC7473PL toxinprotein. Possible additional polypeptides for such a composition includean insect inhibitory protein and an insect inhibitory dsRNA molecule.One example for the use of such ribonucleotide sequences to controlinsect pests is described in Baum, et al. (U.S. Patent Publication2006/0021087 A1). Such additional polypeptide for the control ofLepidopteran pests may be selected from the group consisting of aninsect inhibitory protein, such as, but not limited to, Cry1A (U.S. Pat.No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (U.S. PatentPublication Ser. No. 10/525,318), Cry1C (U.S. Pat. No. 6,033,874),Cry1D, Cry1Da and variants thereof, Cry1E, Cry1F, and Cry1A/F chimeras(U.S. Pat. Nos. 7,070,982; 6,962,705; and 6,713,063), Cry1G, Cry1H,Cry1I, Cry1J, Cry1K, Cry1L, Cry1-type chimeras such as, but not limitedto, TIC836, TIC860, TIC867, TIC869, and TIC1100 (InternationalApplication Publication WO2016/061391 (A2)), TIC2160 (InternationalApplication Publication WO2016/061392(A2)), Cry2A, Cry2Ab (U.S. Pat. No.7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A,Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab,VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045 (U.S. PatentPublication 2013-0117884 A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97,AXMI-102, AXMI-112, AXMI-117, AXMI-100 (U.S. Patent Publication2013-0310543 A1), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication2013-0104259 A1), AXMI-134 (U.S. Patent Publication 2013-0167264 A1),AXMI-150 (U.S. Patent Publication 2010-0160231 A1), AXMI-184 (U.S.Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207,AXMI-209 (U.S. Patent Publication 2011-0030096 A1), AXMI-218, AXMI-220(U.S. Patent Publication 2014-0245491 A1), AXMI-221z, AXMI-222z,AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 2014-0196175A1), AXMI-238 (U.S. Patent Publication 2014-0033363 A1), AXMI-270 (U.S.Patent Publication 2014-0223598 A1), AXMI-345 (U.S. Patent Publication2014-0373195 A1), AXMI-335 (International Application PublicationWO2013/134523(A2)), DIG-3 (U.S. Patent Publication 2013-0219570 A1),DIG-5 (U.S. Patent Publication 2010-0317569 A1), DIG-11 (U.S. PatentPublication 2010-0319093 A1), AfIP-1A and derivatives thereof (U.S.Patent Publication 2014-0033361 A1), AfIP-1B and derivatives thereof(U.S. Patent Publication 2014-0033361 A1), PIP-1APIP-1B (U.S. PatentPublication 2014-0007292 A1), PSEEN3174 (U.S. Patent Publication2014-0007292 A1), AECFG-592740 (U.S. Patent Publication 2014-0007292A1), Pput_1063 (U.S. Patent Publication 2014-0007292 A1), DIG-657(International Application Publication WO2015/195594(A2)), Pput_1064(U.S. Patent Publication 2014-0007292 A1), GS-135 and derivativesthereof (U.S. Patent Publication 2012-0233726 A1), GS153 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS154 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS155 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2012-0167259A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2012-0047606 A1, SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Patent Publication 2011-0154536 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2011-0112013A1, SEQ ID NO:2 and 4 and derivatives thereof as described in U.S.Patent Publication 2010-0192256 A1, SEQ ID NO:2 and derivatives thereofas described in U.S. Patent Publication 2010-0077507 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2010-0077508A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2009-0313721 A1, SEQ ID NO:2 or 4 and derivatives thereof asdescribed in U.S. Patent Publication 2010-0269221 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Pat. No. 7,772,465 (B2),CF161_0085 and derivatives thereof as described in WO2014/008054 A2,Lepidopteran toxic proteins and their derivatives as described in USPatent Publications US2008-0172762 A1, US2011-0055968 A1, andUS2012-0117690 A1; SEQ ID NO:2 and derivatives thereof as described inU.S. Pat. No. 7,510,878(B2), SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Pat. No. 7,812,129(B1); and the like.

In other embodiments, such composition/formulation can further compriseat least one additional polypeptide that exhibits insect inhibitoryactivity to an insect that is not inhibited by an otherwise insectinhibitory protein of the present invention to expand the spectrum ofinsect inhibition obtained. For example, for the control of Hemipteranpests, combinations of insect inhibitory proteins of the presentinvention can be used with Hemipteran-active proteins such as TIC1415(US Patent Publication 2013-0097735 A1), TIC807 (U.S. Pat. No.8,609,936), TIC834 (U.S. Patent Publication 2013-0269060 A1), AXMI-036(U.S. Patent Publication 2010-0137216 A1), and AXMI-171 (U.S. PatentPublication 2013-0055469 A1). Further a polypeptide for the control ofColeopteran pests may be selected from the group consisting of an insectinhibitory protein, such as, but not limited to, Cry3Bb (U.S. Pat. No.6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34/35, 5307,AXMI134 (U.S. Patent Publication 2013-0167264 A1) AXMI-184 (U.S. PatentPublication 2010-0004176 A1), AXMI-205 (U.S. Patent Publication2014-0298538 A1), AXMI-207 (U.S. Patent Publication 2013-0303440 A1),AXMI-218, AXMI-220 (U.S. Patent Publication 20140245491A1), AXMI-221z,AXMI-223z (U.S. Patent Publication 2014-0196175 A1), AXMI-279 (U.S.Patent Publication 2014-0223599 A1), AXMI-R1 and variants thereof (U.S.Patent Publication 2010-0197592 A1, TIC407, TIC417, TIC431, TIC807,TIC853, TIC901, TIC1201, TIC3131, DIG-10 (U.S. Patent Publication2010-0319092 A1), eHIPs (U.S. Patent Application Publication No.2010/0017914), IP3 and variants thereof (U.S. Patent Publication2012-0210462 A1), and ω-Hexatoxin-Hv1a (U.S. Patent ApplicationPublication US2014-0366227 A1).

Additional polypeptides for the control of Coleopteran, Lepidopteran,and Hemipteran insect pests can be found on the Bacillus thuringiensistoxin nomenclature website maintained by Neil Crickmore (on the worldwide web at btnomenclature.info).

The possibility for insects to develop resistance to certaininsecticides has been documented in the art. One insect resistancemanagement strategy is to employ transgenic crops that express twodistinct insect inhibitory agents that operate through different modesof action. Therefore, any insects with resistance to either one of theinsect inhibitory agents can be controlled by the other insectinhibitory agent. Another insect resistance management strategy employsthe use of plants that are not protected to the targeted Lepidopteranpest species to provide a refuge for such unprotected plants. Oneparticular example is described in U.S. Pat. No. 6,551,962, which isincorporated by reference in its entirety.

Other embodiments such as topically applied pesticidal chemistries thatare designed for controlling pests that are also controlled by theproteins disclosed herein to be used with proteins in seed treatments,spray on, drip on, or wipe on formulations can be applied directly tothe soil (a soil drench), applied to growing plants expressing theproteins disclosed herein, or formulated to be applied to seedcontaining one or more transgenes encoding one or more of the proteinsdisclosed. Such formulations for use in seed treatments can be appliedwith various stickers and tackifiers known in the art. Such formulationscan contain pesticides that are synergistic in mode of action with theproteins disclosed, so that the formulation pesticides act through adifferent mode of action to control the same or similar pests that canbe controlled by the proteins disclosed, or that such pesticides act tocontrol pests within a broader host range or plant pest species that arenot effectively controlled by the TIC6757, TIC6757PL, TIC7472,TIC7472PL, TIC7473, or TIC7473PL pesticidal proteins.

The aforementioned composition/formulation can further comprise anagriculturally-acceptable carrier, such as a bait, a powder, dust,pellet, granule, spray, emulsion, a colloidal suspension, an aqueoussolution, a Bacillus spore/crystal preparation, a seed treatment, arecombinant plant cell/plant tissue/seed/plant transformed to expressone or more of the proteins, or bacterium transformed to express one ormore of the proteins. Depending on the level of insect inhibitory orinsecticidal inhibition inherent in the recombinant polypeptide and thelevel of formulation to be applied to a plant or diet assay, thecomposition/formulation can include various by weight amounts of therecombinant polypeptide, e.g. from 0.0001% to 0.001% to 0.01% to 1% to99% by weight of the recombinant polypeptide.

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, specific structural and functionaldetails disclosed herein are not to be interpreted as limiting. Itshould be understood that the entire disclosure of each reference citedherein is incorporated within the disclosure of this application.

EXAMPLES Example 1 Discovery, Cloning, and Expression of TIC6757

Sequences encoding three novel Paenibacillus popilliae pesticidalproteins were identified, cloned, sequence confirmed, and tested ininsect bioassay. The pesticidal proteins, TIC6757, TIC7472, and TIC7473,isolated from the Paenibacillus popilliae strains DSC004343, DSC007648,and DSC008493, respectively, represent novel Vip3C-like proteins.Distant-related sequences to TIC6757, TIC7472, and TIC7473 are Vip3Ca2(at 83.7% identity, the closest known relative), Vip3Aa1 (66.75%identity), and a Vip3B-like protein (60.93% identity). The distinctiveand unique quality of TIC6757, TIC7472, and TIC7473 indicates that thesepesticidal proteins likely have a novel mode of action (MOA).

Polymerase chain reaction (PCR) primers were designed to amplify a fulllength copy of the coding region for TIC6757, TIC7472, and TIC7473 fromtotal genomic DNA isolated from the Paenibacillus popilliae strainsDSC004343, DSC007648, and DSC008493, respectively. The PCR ampliconsalso included the translational initiation and termination codons ofeach coding sequence.

Each of the amplicons were cloned using methods known in the art intotwo different Bt expression vectors in operable linkage with a Btexpressible promoter. One Bt expression vector comprised a promoter thatis on during sporulation of the bacillus. The other expression vectorcomprised a non-sporulation promoter. In addition, each of the ampliconswere cloned into a vector used for protein expression in Escherichiacoli (E. coli). For isolation of the E. coli expressed proteins, aHistidine tag was operably linked to the expressed coding sequences tofacilitate column purification of the protein. The coding sequences andtheir respective protein sequences used for bacterial expression arepresented in Table 3 below.

TABLE 3 Toxin coding sequences and corresponding protein sequences usedfor expression in Bt and E. coli. DNA Coding Protein Sequence SEQBacterial SEQ ID ID Expression Toxin NO: NO: Host TIC6757 1 2 Bt TIC74727 8 Bt TIC7473 11 12 Bt TIC6757_His 5 6 E. coli TIC7472_His 9 10 E. coliTIC7473_His 13 14 E. coli

Example 2 TIC6757, TIC7472, and TIC7473 Demonstrates LepidopteranActivity in Insect Bioassay

The pesticidal proteins TIC6757, TIC7472, and TIC7473 were expressed inBt and E. coli and assayed for toxicity to various species ofLepidoptera, Coleoptera, and Hemiptera. Preparations of each toxin fromBt were assayed against the Lepidopteran species Beet armyworm (BAW,Spodoptera exigua), Black cutworm (BCW, Agrotis Corn earworm (CEW,Helicoverpa zea), Cotton leaf worm (CLW, Alabama argillacea),Diamondback moth (DBM, Plutella xylostella), European corn borer (ECB,Ostrinia nubilalis), Fall armyworm (FAW, Spodoptera frugiperda), Cry1Fa1resistant Fall armyworm (FAWR1, Spodoptera frugiperda), Americanbollworm (AWB, Helicoverpa armigera), Pink bollworm (PBW, Pectinophoragossypiella), Southern armyworm (SAW, Spodoptera eridania), Soybeanlooper (SBL, Chrysodeixis includens), Spotted bollworm (SBW, Eariasvittella), Southwestern corn borer (SWCB, Diatraea grandiosella),Tobacco budworm (TBW, Heliothis virescens), Tobacco cutworm (TCW,Spodoptera litura, also known as cluster caterpillar), and Velvet beancaterpillar (VBW, Anticarsia gemmatalis); the coleopteran speciesColorado potato beetle (CPB, Leptinotarsa decemlineata), Western CornRootworm (WCB, Diabrotica virgifera virgifera); and the hemipteranspecies Tarnished plant bug (TPB, Lygus lineolaris), Western tarnishedplant bug (WTP, Lygus hesperus), Neotropical Brown Stink Bug (NBSB,Euschistus heros), and Green Stink Bug (GSB, Nezara viridula).

Bioactivity of the pesticidal proteins TIC6757, TIC7472, and TIC7473 wasevaluated by producing the protein in either an E. coli or Bt expressionhost. In the case of the Bt host, a Bt strain expressing TIC6757,TIC7472, or TIC7473 was grown for twenty four (24) hours and then theculture was added to insect diet. Mortality and stunting were evaluatedby comparing the growth and development of insects on a diet with aculture from the Bt strain expressing TIC6757, TIC7472, or TIC7473 toinsects on a diet with an untreated control culture. The E. coli strainsexpressing TIC6757, TIC7472, or TIC7473 were treated in a similar mannerand were also provided in an insect diet. The bioassay activity observedfor each protein from either the Bt or E. coli preparation or bothpreparations is presented in Tables 4 and 5 below, wherein “+” indicatesactivity and “NT” indicates the toxin was not assayed against thatspecific insect pest.

TABLE 4 Bioassay activity of TIC6757, TIC7472, and TIC7473 againstinsect pests. Toxin BAW BCW CEW CLW DBM ECB FAW FAWR1 AWB PBW SAW SBLTIC6757 + + + + + + + + + + + TIC7472 NT NT + NT NT NT + NT NT NT + +TIC7473 NT NT + NT NT NT + NT NT NT + +

TABLE 5 Bioassay activity of TIC6757, TIC7472, and TIC7473 againstinsect pests. Toxin SBW SWCB TBW TCW VBC CPB WCR TPB WTP NBSB SGBTIC6757 + + + + + TIC7472 NT + NT NT NT NT NT TIC7473 NT + NT NT NT NTNT

As can be seen in Tables 4 and 5 above, the insect toxin TIC6757demonstrated activity against many Lepidopteran insect pests (BAW, BCW,CEW, CLW, DBM, ECB, FAW, FAWR1, AWB, SAW, SBL, SBW, SWCB, TBW, TCW, andVBC). Activity was observed for most of the pests assayed againstTIC7472 and TIC7473 (CEW, FAW, SAW, SBL, SWCB).

Example 3 Assay of TIC6757PL Activity Against Lepidopteran Pests inStably Transformed Corn Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC6757PLpesticidal protein were cloned using methods known in the art. Theresulting vectors were used to stably transform corn plants. Tissueswere harvested from the transformants and used in insect bioassayagainst various Lepidopteran insect pests.

Synthetic coding sequences were constructed for use in expression of theencoded protein in plants, cloned into a binary plant transformationvector, and used to transform corn plant cells. The synthetic sequenceswere synthesized, according to methods generally described in U.S. Pat.No. 5,500,365, to avoid certain inimical problem sequences such as ATTTAand A/T rich plant polyadenylation sequences while preserving the aminoacid sequence of the native Paenibacillus protein. The synthetic codingsequences encoded a TIC6757PL protein which comprises an additionalalanine residue immediately following the initiating methionine relativeto TIC6757 protein. For plastid targeted protein, the syntheticTIC6757PL pesticidal protein coding sequence was operably linked inframe with a chloroplast targeting signal peptide coding sequence. Theresulting plant transformation vectors comprised a first transgenecassette for expression of the TIC6757PL pesticidal protein whichcomprised a constitutive promoter, operably linked 5′ to a leader,operably linked 5′ to an intron, operably linked 5′ to a syntheticcoding sequence encoding a plastid targeted or untargeted TIC6757PLprotein, which was in turn operably linked 5′ to a 3′ UTR; and a secondtransgene cassette for the selection of transformed plant cells usingglyphosate selection. The synthetic coding sequence for the TIC6757PLpesticidal protein is presented as SEQ ID NO:3 and encodes the proteinpresented as SEQ ID NO:4.

Corn plants were transformed with four different binary transformationvectors as described above using an Agrobacterium-mediatedtransformation method. Binary plant transformation vector Constructs 1and 3 comprised a coding sequence encoding a plastid targeted TIC6757PLprotein, while Constructs 2 and 4 comprised a coding sequence encoding anon-targeted TIC6757PL protein. The transformed cells were induced toform plants by methods known in the art. Bioassays using plant leafdisks were performed analogous to those described in U.S. Pat. No.8,344,207. A single freshly hatched neonate larvae less than one day oldwas placed on each leaf disc sample and allowed to feed forapproximately four days. A non-transformed corn plant was used to obtaintissue to be used as a negative control. Multiple transformation R₀single-copy insertion events from each binary vector were assessedagainst Black cutworm (BCW, Agrotis ipsilon), Corn earworm (CEW,Helicoverpa zea), Fall armyworm (FAW, Spodoptera frugiperda), andSouthwestern Corn Borer (SWCB, Diatraea grandiosella).

Transformed R₀ plants expressing TIC6757PL were highly efficacious(defined as having less than or equal to seventeen point five percentleaf damage with one hundred percent mortality) against all four insectpests assayed as shown in Table 6. High penetrance (indicated by “(H)”)is defined as greater than fifty percent of the assayed events for eachconstruct having less than or equal to seventeen point five percent leafdamage with one hundred percent mortality. Low penetrance (indicated by“(L)”) is defined as less than or equal to fifty percent of the assayedevents for each construct having less than or equal to seventeen pointfive percent leaf damage with one hundred percent mortality.

TABLE 6 Number of Events Expressing TIC6757 with ≤ 17.5% Leaf Damagewith One Hundred Percent Mortality and Penetrance. Total Number ofEvents with ≤ 17.5% Number Leaf Damage and 100% mortality of(penetrance) Construct Events BCW CEW FAW SWC Construct 1 22 17 (H) 18(H) 18 (H) 11 (L) Construct 2 20 14 (H) 14 (H) 14 (H)  4 (L) Construct 319 17 (H) 17 (H) 17 (H) 17 (H) Construct 4 20 16 (H) 16 (H) 15 (H)  7(L)

Selected R₀ events derived from R₀ Construct 1 (plastid targeted) andConstruct 2 plastid untargeted) were allowed to self-pollinate,producing F₁ progeny. Several heterozygous F₁ progeny plants from eachR₀ event were selected for leaf disc bioassay and assayed against Blackcutworm (BCW, Agrotis ipsilon), Corn earworm (CEW, Helicoverpa zea),Fall armyworm (FAW, Spodoptera frugiperda), and Southwestern Corn Borer(SWCB, Diatraea grandiosella). Table 7 below shows the mean percent leafdamage and mean mortality for each plant derived from eachconstruct/event. The F₁ progeny plants are referenced with respect tothe R₀ event. For example “Event-1_1” is the first heterozygous F₁progeny plant derived from Event-1 and “Event-1_2” is the firstheterozygous F₁ progeny plant derived from Event-1. “N” represents thenumber of samples from each plant used in assay. As can be seen inTables 7 and 8, most plants derived from each R₀ event demonstrated nomore than five percent leaf damage and one hundred percent mortalityagainst BCW, CEW, and FAW. With respect to SWCB, multiple plants derivedfrom each R₀ event demonstrated less than ten percent leaf damage andgreater than fifty percent mortality in assay.

TABLE 7 Mean Percent Leaf Damage and Mortality in F₁ Progeny Derivedfrom Selected R₀ events Expressing TIC6757PL. BCW CEW Mean % Mean Mean %Mean Leaf Mortal- Leaf Mortal- Construct Event_Plant N Damage ity Damageity Construct 1 Event-1_1 3 5.00 100.00 5.00 100.00 Construct 1Event-1_2 3 5.00 100.00 5.00 100.00 Construct 1 Event-1_3 3 5.00 100.005.00 100.00 Construct 1 Event-1_4 3 5.00 100.00 6.65 100.00 Construct 1Event-2_1 3 5.00 100.00 5.00 100.00 Construct 1 Event-2_2 3 NT NT 7.50100.00 Construct 1 Event-2_3 3 NT NT 8.35 100.00 Construct 2 Event-3_1 35.00 100.00 5.00 100.00 Construct 2 Event-3_2 3 5.00 100.00 5.00 100.00Construct 2 Event-4_1 3 5.00 100.00 5.00 100.00 Construct 2 Event-4_2 35.00 100.00 5.00 100.00 Construct 2 Event-4_3 3 6.65 66.67 5.00 100.00Construct 2 Event-4_4 3 6.65 66.67 5.00 100.00 Construct 2 Event-4_5 320.00 33.33 10.00 100.00 Construct 2 Event-5_1 3 5.00 100.00 5.00 100.00Construct 2 Event-5_2 3 5.00 100.00 5.00 100.00 Construct 2 Event-5_3 35.00 100.00 5.00 100.00 NONE Negative 3 55.00 0.00 55.00 0.00 Control

TABLE 8 Mean Percent Leaf Damage and Mortality in F₁ Progeny Derivedfrom Selected R₀ events Expressing TIC6757PL. FAW SWCB Mean % Mean Mean% Mean Leaf Mortal- Leaf Mortal- Construct Event_Plant N Damage ityDamage ity Construct 1 Event-1_1 3 5.00 100.00 6.65 66.67 Construct 1Event-1_2 3 5.00 100.00 6.65 66.67 Construct 1 Event-1_3 3 5.00 100.007.50 50.00 Construct 1 Event-1_4 3 5.00 100.00 8.35 66.67 Construct 1Event-2_1 3 5.00 100.00 5.00 50.00 Construct 1 Event-2_2 3 5.00 100.005.00 50.00 Construct 1 Event-2_3 3 5.00 100.00 6.65 66.67 Construct 2Event-3_1 3 5.00 100.00 5.00 100.00 Construct 2 Event-3_2 3 5.00 100.0015.00 50.00 Construct 2 Event-4_1 3 5.00 100.00 12.50 0.00 Construct 2Event-4_2 3 5.00 100.00 40.00 100.00 Construct 2 Event-4_3 3 5.00 100.0048.35 0.00 Construct 2 Event-4_4 3 5.00 100.00 55.00 0.00 Construct 2Event-4_5 3 5.00 100.00 55.00 0.00 Construct 2 Event-5_1 3 5.00 100.005.00 100.00 Construct 2 Event-5_2 3 5.00 100.00 6.65 66.67 Construct 2Event-5_3 3 5.00 100.00 8.35 0.00 NONE Negative 3 55.00 0.00 51.65 0.00Control

Selected R₀ events derived from Construct 3 (plastid targeted) andConstruct 4 (untargeted) were allowed to self-pollinate producing F₁progeny. A heterozygous F₁ progeny plant from each R₀ event was selectedfor leaf disc bioassay and assayed against Western bean cutworm (WBC,Striacosta albicosta). Table 9 shows the mean percent leaf damage andmean percent mortality of the F₁ progeny plant from each R₀ event andthe negative control. “N” represents the number of samples from eachplant used in assay.

TABLE 9 Mean Percent Leaf Damage and Mean Percent Mortality in F₁Progeny Derived from Selected R₀ events Expressing TIC6757PL. Mean %Leaf Mean Construct Event N Damage Mortality Construct 3 Event-6_1 45.00 100.00 Construct 3 Event-7_1 4 5.00 100.00 Construct 3 Event-8_1 45.00 100.00 Construct 3 Event-9_1 4 5.00 100.00 Construct 3 Event-10_1 45.00 100.00 Construct 3 Event-11_1 4 5.00 100.00 Construct 3 Event-12_14 5.00 100.00 Construct 3 Event-13_1 4 5.00 100.00 Construct 3Event-14_1 4 5.00 100.00 Construct 3 Event-15_1 4 27.50 50.00 Construct4 Event-16_1 4 5.00 100.00 Construct 4 Event-17_1 4 5.00 100.00Construct 4 Event-18_1 4 5.00 100.00 Negative 4 45.00 0.00 Control

As can be seen in Table 9 above, all but one F₁ progeny plant from eachR₀ event assayed against WBC demonstrated no more than five percent leafdamage and one hundred percent mortality.

Seedlings derived from selected heterozygous F₁ progeny plantstransformed with Construct 3 (plastid targeted) and Construct 4(untargeted) were assayed for resistance against Black cutworm (BCW,Agrotis ipsilon). F₁ progeny seeds, as well as non-transformed seed(negative control), were planted in pots. After eight days when theseedlings were emerging from the soil, each plant was infested withthree, third instar BCW. Fourteen days after infestation the plants wereinspected to count the number of plants that were cut down by BCW. Sixtyeight F₁ progeny plants derived from ten different R₀ events transformedwith Construct 3 and ten F₁ progeny plants derived from four differentR₀ events transformed with Construct 4 were used in the assay. Fifteennegative control plants were also used in the assay. After inspection ofthe plants, it was observed that eighty percent of the negative controlswere cut down by BCW while zero percent of the F₁ progeny plantstransformed with either Construct 3 and Construct 4 demonstratedcutting.

The forgoing demonstrates that transformed corn plants expressingTIC6757PL provide superior resistance to Lepidopteran insect pests, inparticular Black cutworm (Agrotis ipsilon), Corn earworm (Helicoverpazea), Fall armyworm (Spodoptera frugiperda), Southwestern Corn Borer(Diatraea grandiosella), and Western bean cutworm (Striacostaalbicosta).

Example 4 Assay of TIC6757PL Activity Against Lepidopteran Pests inStably Transformed Soybean Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC6757PLpesticidal protein were cloned using methods known in the art. Theresulting vectors were used to stably transform soybean plants. Tissueswere harvested from the transformants and used in insect bioassayagainst various Lepidopteran insect pests.

The synthetic coding sequence designed for plant expression as describedin Example 3 above was cloned into binary plant transformation vectors,and used to transform soybean plant cells. Binary vectors comprisingplastid targeted and untargeted TIC6757PL coding sequences wereconstructed using methods known in the art. The resulting planttransformation vectors comprised a first transgene cassette forexpression of the TIC6757PL pesticidal protein which comprised aconstitutive promoter, operably linked 5′ to a leader, operably linked5′ to a synthetic coding sequence encoding a plastid targeted oruntargeted TIC6757PL protein, which was in turn operably linked 5′ to a3′ UTR and; a second transgene cassette for the selection of transformedplant cells using spectinomycin selection. Constructs 1, 3 and 5comprised a coding sequence encoding an untargeted TIC6757PL pesticidalprotein. Constructs 2, 4 and 6 comprised a coding sequence encoding aplastid targeted TIC6757PL protein.

The transformed soybean cells were induced to form plants by methodsknown in the art. Bioassays using plant leaf disks were performedanalogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed soybean plant was used to obtain tissue to be used as anegative control. Multiple transformation events from each binary vectorwere assessed against Southern armyworm (SAW, Spodoptera eridania),Soybean looper (SBL, Chrysodeixis includens), and Soybean podworm (SPW,Helicoverpa zea).

Transformed R₀ soybean plants expressing TIC6757PL were highlyefficacious (defined as having less than or equal to twenty percent leafdamage) against SAW, SBL, and SPW as shown in Table 10. High penetrance(indicated by “(H)”) is defined as greater than fifty percent of theassayed events for each construct having less than or equal to twentypercent leaf damage. Low penetrance (indicated by “(L)”) is defined asless than or equal to fifty percent of the assayed events for eachconstruct having less than or equal to twenty percent leaf damage.

TABLE 10 Number of Events Expressing TIC6757PL with ≤ 20% Leaf Damageand Penetrance. Number of Events with ≤ 20% Total Leaf Damage Number of(Penetrance) Construct Events SAW SBL SPW Construct 1 15 14 (H) 14 (H)12 (H) Construct 2 15  5 (L)  3 (L)  8 (H) Construct 3 15 12 (H) 13 (H)13 (H) Construct 4 15 15 (H) 15 (H) 15 (H) Construct 5 15 14 (H) 13 (H)14 (H) Construct 6 15 15 (H) 15 (H) 15 (H)

Selected R₀ transgenic soybean plants expressing TIC6757PL protein toxinderived from transformation of Constructs 3, 4, 5, and 6 were allowed toself-pollinate and produce R₁ seed. The R₁ seed was allowed to germinateproducing R₁ plants. R₁ plants homozygous for the TIC6757PL expressioncassette were selected for leaf disc bioassay against Southern armyworm(SAW, Spodoptera eridania), Soybean looper (SBL, Chrysodeixisincludens), Soybean podworm (SPW, Helicoverpa zea), and Velvet beancaterpillar (VBW, Anticarsia gemmatalis). Tables 11 and 12 show the meanpercent leaf damage demonstrated by each insect for each R₁ progenyplant and the negative control, variety A3555. Tables 11 and 12 alsoshow the standard error mean (SEM) percent leaf damage demonstrated byeach insect for each event assayed relative to the negative control. “N”represents the number of samples from each plant used in assay. “SEM”represents the standard error of the mean percent damage.

TABLE 11 Mean Percent Leaf Damage for R₁ Soybean Plants ExpressingTIC6757PL. SAW SBL Number Mean Mean Number of % % of Plants/ Dam- Dam-Construct Events Event N age SEM N age SEM Construct 3 5 6 4 0.37 0.30 41.91 0.72 Construct 4 8 6 4 0.31 0.25 4 1.25 0.34 Construct 5 8 6 4 0.020.02 4 0.75 0.35 Construct 6 8 6 4 0.76 0.34 4 0.97 0.35 NegativeVariety 8 4 87.93 9.74 4 79.44 12.44 Control A3555

TABLE 12 Mean Percent Leaf Damage for R₁ Soybean Plants ExpressingTIC6757PL. SPW VBC Number Mean Mean Number of % % of Plants/ Dam- Dam-Construct Events Event N age SEM N age SEM Construct 3 5 6 4 16.32 3.834 1.89 0.60 Construct 4 8 6 4 2.25 0.30 4 0.96 0.31 Construct 5 8 6 42.40 0.50 4 0.51 0.25 Construct 6 8 6 4 3.65 0.53 4 0.71 0.32 NegativeVariety 8 4 97.25 1.09 4 88.88 10.30 Control A3555

As can be seen in Tables 11 and 12, R₁ soybean plants expressingTIC6757PL toxin protein provide superior resistance to SAW, SBL, SPW,and VBC. With respect to SAW, all four events demonstrated less than one(1) percent leaf damage while the negative control had approximatelyeighty-eight (88) percent leaf damage. With respect to SBL, all four (4)events demonstrated less than two (2) percent leaf damage while thecontrol had approximately eighty (80) percent leaf damage. With respectto SPW, three of the four events demonstrated less than four (4) percentleaf damage while the control had approximately ninety-seven (97)percent leaf damage. With respect to VBC, three of the eventsdemonstrated less than one (1) percent leaf damage and one eventdemonstrated less than two (2) percent leaf damage, while the negativecontrol had close to eighty-nine (89) percent leaf damage.

The forgoing demonstrates that transformed soybean plants expressingTIC6757PL provide superior resistance to Lepidopteran insects, inparticular Southern armyworm (Spodoptera eridania), Soybean looper(Chrysodeixis includens), Soybean podworm (Helicoverpa zea), and Velvetbean caterpillar (Anticarsia gemmatalis).

Example 5 Assay of TIC6757PL Activity Against Lepidopteran Pests inStably Transformed Cotton Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC6757PLpesticidal protein were cloned using methods known in the art. Theresulting vectors were used to stably transform cotton plants. Tissueswere harvested from the transformants and used in insect bioassayagainst various Lepidopteran insect pests.

The synthetic coding sequence designed for plant expression as describedin Example 3 above was cloned into binary plant transformation vectors,and used to transform cotton plant cells. Binary vectors comprisingplastid targeted and untargeted TIC6757PL coding sequences wereconstructed using methods known in the art. The resulting planttransformation vectors comprised a first transgene cassette forexpression of the TIC6757PL pesticidal protein which comprised aconstitutive promoter, operably linked 5′ to a leader, operably linked5′ to a synthetic coding sequence encoding a plastid targeted oruntargeted TIC6757PL protein, which was in turn operably linked 5′ to a3′ UTR and; a second transgene cassette for the selection of transformedplant cells using spectinomycin selection.

The transformed cotton cells were induced to form plants by methodsknown in the art. Bioassays using plant leaf disks were performedanalogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed cotton plant was used to obtain tissue to be used as anegative control. Multiple transformation events from each binary vectorwere assessed against Southern armyworm Cotton bollworm (CBW,Helicoverpa zea), Fall armyworm (FAW, Spodoptera frugiperda), Soybeanlooper (SBL, Chrysodeixis includens), and Tobacco budworm (TBW,Heliothis virescens).

Transformed R₀ cotton plants expressing TIC6757PL were highlyefficacious (defined as having less than or equal to ten percent leafdamage) against CBW, FAW, SBL and TBW as shown in Table 13. Highpenetrance (as indicated by “(H)”) is defined as greater than fiftypercent of the assayed events for each construct having less than orequal to ten percent leaf damage. Low penetrance (as indicated by “(L)”)is defined as less than or equal to fifty percent of the assayed eventsfor each construct having less than or equal to ten percent leaf damage.

TABLE 13 Number of Events Expressing TIC6757PL with ≤ 10% Leaf Damageand Penetrance. Number of Events with ≤ 10% Leaf Damage/Number eventsassayed (Penetrance) Construct CBW FAW SBL TBW Construct 1 22/25 (H)21/24 (H) 21/25 (H) 21/25 (H) Construct 2 12/15 (H)  6/15 (L) 13/15 (H)13/15 (H) Construct 3  7/13 (H)  8/14 (H)  4/13 (L)  6/14 (L) Construct4 11/14 (H)  8/14 (H)  9/14 (H) 10/14 (H) Construct 5 20/25 (H) 19/23(H) 20/24 (H) 19/23 (H) Construct 6  6/7 (H)  7/7 (H)  7/7 (H)  6/7 (H)Construct 7 22/25 (H) 22/25 (H) 22/25 (H) 22/25 (H)

Example 6 Assay of TIC7472PL and TIC7473PL Activity Against LepidopteranPests in Stably Transformed Corn Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC7472PL orTIC7473PL pesticidal protein are cloned using methods known in the art.The resulting vectors are used to stably transform corn plants. Tissuesare harvested from the transformants and used in insect bioassay againstvarious Lepidopteran insect pests.

Synthetic coding sequences are constructed for use in expression of theencoded protein in plants, cloned into a binary plant transformationvector, and used to transform corn plant cells. The synthetic sequencesare synthesized according to methods generally described in U.S. Pat.No. 5,500,365, avoiding certain inimical problem sequences such as ATTTAand A/T rich plant polyadenylation sequences while preserving the aminoacid sequence of the native Paenibacillus protein. The synthetic codingsequences encode a TIC7472PL and TIC7473PL protein, which comprise anadditional alanine residue immediately following the initiatingmethionine relative to the TIC7472 and TIC7473 protein. For plastidtargeted protein, the synthetic TIC7472PL or TIC7473PL pesticidalprotein coding sequence is operably linked in frame with a chloroplasttargeting signal peptide coding sequence. The resulting planttransformation vectors comprise a first transgene cassette forexpression of the TIC7472PL or TIC7473PL pesticidal protein whichcomprise a constitutive promoter, operably linked 5′ to a leader,operably linked 5′ to an intron, operably linked 5′ to a syntheticcoding sequence encoding a plastid targeted or untargeted TIC7472PL orTIC7473PL protein, which is in turn operably linked 5′ to a 3′ UTR; anda second transgene cassette for the selection of transformed plant cellsusing glyphosate selection. The synthetic coding sequence for theTIC7472PL pesticidal protein is presented as SEQ ID NO:15 and encodesthe protein presented as SEQ ID NO:16. The synthetic coding sequence forthe TIC7473PL pesticidal protein is presented as SEQ ID NO:17 andencodes the protein presented as SEQ ID NO:18.

Corn plants are transformed with the binary transformation vectorsdescribed above using an Agrobacterium-mediated transformation method.The transformed cells are induced to form plants by methods known in theart. Bioassays using plant leaf disks are performed analogous to thosedescribed in U.S. Pat. No. 8,344,207. A non-transformed corn plant isused to obtain tissue to be used as a negative control. Multipletransformation events from each binary vector were assessed againstBlack cutworm (BCW, Agrotis ipsilon), Corn earworm (CEW, Helicoverpazea), Fall armyworm (FAW, Spodoptera frugiperda), and Southwestern CornBorer (SWCB, Diatraea grandiosella), as well as other Lepidoteran insectpests.

The insect pests are observed for mortality and stunting caused byingestion of the presented leaf discs expressing TIC7472PL or TIC7473PLand compared to leaf discs derived from non-transformed corn plants.

Example 7 Assay of TIC6757PL Activity Against Lepidopteran Pests inStably Transformed Soybean and Cotton Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC7472PL orTIC7473PL pesticidal protein are cloned using methods known in the art.The resulting vectors are used to stably transform soybean and cottonplants. Tissues are harvested from the transformants and used in insectbioassay against various Lepidopteran insect pests.

The synthetic coding sequences designed for plant expression asdescribed in Example 6 above are cloned into binary plant transformationvectors, and used to transform soybean or cotton plant cells. Binaryvectors comprising plastid targeted and untargeted TIC7472PL orTIC7473PL coding sequences are constructed using methods known in theart. The resulting plant transformation vectors comprise a firsttransgene cassette for expression of the TIC7472PL or TIC7473PLpesticidal protein which comprise a constitutive promoter, operablylinked 5′ to a leader, operably linked 5′ to a synthetic coding sequenceencoding a plastid targeted or untargeted TIC7472PL or TIC7473PLprotein, which is in turn operably linked 5′ to a 3′ UTR and; a secondtransgene cassette for the selection of transformed plant cells usingspectinomycin selection. Constructs 1, 2 and 7 comprised a cloningsequence encoding an untargeted TIC6757PL pesticidal protein. Constructs3, 4, 5 and 6 comprised a coding sequence encoding a targeted TIC6757PLpesticidal protein.

The transformed soybean or cotton cells are induced to form plants bymethods known in the art. Bioassays using plant leaf disks are performedanalogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed soybean or cotton plant is used to obtain tissue to beused as a negative control. Multiple transformation events from eachbinary vector are assessed against Southern armyworm (SAW, Spodopteraeridania), Soybean looper (SBL, Chrysodeixis includens), Soybean podworm(SPW, Helicoverpa zea) Fall armyworm (FAW, Spodoptera frugiperda),Soybean looper (SBL, Chrysodeixis includens), Tobacco budworm (Heliothisvirescens), Cotton bollworm (CBW, Helicoverpa zea), and Velvet beancaterpillar (VBW, Anticarsia gemmatalis) as well as other Lepidoteraninsect pests. The insect pests are observed for mortality and stuntingcaused by ingestion of the presented leaf discs expressing TIC7472PL orTIC7473PL and compared to leaf discs derived from non-transformedsoybean or cotton plants.

All of the compositions disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions of this invention have been describedin terms of the foregoing illustrative embodiments, it will be apparentto those of skill in the art that variations, changes, modifications,and alterations may be applied to the composition described herein,without departing from the true concept, spirit, and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope, andconcept of the invention as defined by the appended claims.

All publications and published patent documents cited in thespecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

What is claimed is:
 1. A recombinant nucleic acid molecule comprising aheterologous promoter operably linked to a polynucleotide segmentencoding a protein wherein said protein comprises an amino acid sequencehaving the amino acid sequence as set forth in SEQ ID NO:2.
 2. A plantcell expressing the recombinant nucleic acid molecule of claim 1,wherein said plant cell produces a protein or protein fragment encodedby said recombinant nucleic acid molecule.
 3. The plant cell of claim 2,wherein said plant cell is a dicotyledonous or a monocotyledonous plantcell.
 4. The plant cell of claim 3, wherein said plant cell is selectedfrom the group consisting of alfalfa, banana, barley, bean, broccoli,cabbage, carrot, cassava, castor, cauliflower, celery, chickpea, Chinesecabbage, coconut, coffee, corn, clover, cotton, cucumber, Douglas fir,eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblollypine, millets, melons, nut, oat, olive, onion, palm, pasture grass, pea,peanut, pepper, pigeon pea, potato, poplar, pumpkin, Radiata pine,radish, rapeseed, rice, rye, safflower, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetcorn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,triticale, turf grass, watermelon, and wheat plant cell.
 5. A host cellexpressing the recombinant nucleic acid molecule of claim 1, whereinsaid host cell is selected from the group consisting of a bacterial celland a plant cell.
 6. The host cell of claim 5, wherein said host cell isfrom a genus of bacteria selected from the group consisting of:Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia,Pseudomonas, Klebsiella, Pantoea, and Erwinia.
 7. The host cell of claim6, wherein said Bacillus species is Bacillus cereus or Bacillusthuringiensis, said Brevibacillus species is Brevibacilluslaterosperous, and said Escherichia species is Escherichia coli.
 8. Aplant, or part thereof, comprising the recombinant nucleic acid moleculeof claim
 1. 9. The plant, or part thereof, of claim 8, wherein saidplant is a monocot plant or a dicot plant.
 10. The plant of claim 9,wherein said plant is selected from the group consisting of alfalfa,banana, barley, bean, broccoli, cabbage, carrot, cassava, castor,cauliflower, celery, chickpea, Chinese cabbage, coconut, coffee, corn,clover, cotton, cucumber, Douglas fir, eggplant, eucalyptus, flax,garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut,oat, olive, onion, palm, pasture grass, pea, peanut, pepper, pigeon pea,potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rye,safflower, sorghum, Southern pine, soybean, spinach, squash, strawberry,sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato,switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon,and wheat.
 11. A seed of the plant of claim 8, wherein said seedcomprises said recombinant nucleic acid molecule.
 12. A method ofproducing seed, said method comprising: a. planting a first seedaccording to claim 11; b. growing a plant from said seed; and c.harvesting seed from said plant, wherein said harvested seed comprisessaid recombinant nucleic acid molecule.
 13. A commodity product producedfrom the plant, or part thereof, of claim 8, wherein said commodityproduct comprises a detectable amount of said recombinant nucleic acidmolecule or a protein encoded by said recombinant nucleic acid molecule.14. The commodity product of claim 13, selected from the groupconsisting of flakes, cakes, flour, meal, syrup, oil, silage, starch,cereal, juices, concentrates, jams, jellies, marmalades, whole orprocessed seed, lint, fiber, paper, biomass, fuel products, protein,bran, milk, cheese, wine, animal feed, paper, and cream; wherein saidcommodity product is produced from a host cell derived from a plantselected from the group consisting of soybean, rice, wheat, sorghum,pigeon pea, peanut, fruit, melon, and vegetable.